Pikis Andreas, Hess Sonja, Arnold Ingrid, Erni Bernhard, Thompson John
Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research/NIH, Bldg. 30, Convent Drive, Bethesda, MD 20892, USA.
J Biol Chem. 2006 Jun 30;281(26):17900-8. doi: 10.1074/jbc.M601183200. Epub 2006 Apr 24.
Strains of Escherichia coli K12, including MG-1655, accumulate methyl-alpha-D-glucopyranoside via the phosphoenolpyruvate-dependent glucose:phosphotransferase system (IICB(Glc)/IIA(Glc)). High concentrations of intracellular methyl-alpha-D-glucopyranoside 6-phosphate are toxic, and cell growth is prevented. However, transformation of E. coli MG-1655 with a plasmid (pAP1) encoding the gene aglB from Klebsiella pneumoniae resulted in excellent growth of the transformant MG-1655 (pAP1) on the glucose analog. AglB is an unusual NAD+/Mn2+-dependent phospho-alpha-glucosidase that promotes growth of MG-1655 (pAP1) by catalyzing the in vivo hydrolysis of methyl-alpha-D-glucopyranoside 6-phosphate to yield glucose 6-phosphate and methanol. When transformed with plasmid pAP2 encoding the K. pneumoniae genes aglB and aglA (an alpha-glucoside-specific transporter AglA (IICB(Agl))), strain MG-1655 (pAP2) metabolized a variety of other alpha-linked glucosides, including maltitol, isomaltose, and the following five isomers of sucrose: trehalulose alpha(1-->1), turanose alpha(1-->3), maltulose alpha(1-->4), leucrose alpha(1-->5), and palatinose alpha(1-->6). Remarkably, MG-1655 (pAP2) failed to metabolize sucrose alpha(1-->2). The E. coli K12 strain ZSC112L (ptsG::cat manXYZ nagE glk lac) can neither grow on glucose nor transport methyl-alpha-D-glucopyranoside. However, when transformed with pTSGH11 (encoding ptsG) or pAP2, this organism provided membranes that contained either the PtsG or AglA transporters, respectively. In vitro complementation of transporter-specific membranes with purified general phosphotransferase components showed that although PtsG and AglA recognized glucose and methyl-alpha-D-glucopyranoside, only AglA accepted other alpha-D-glucosides as substrates. Complementation experiments also revealed that IIA(Glc) was required for functional activity of both PtsG and AglA transporters. We conclude that AglA, AglB, and IIA(Glc) are necessary and sufficient for growth of E. coli K12 on methyl-alpha-D-glucoside and related alpha-D-glucopyranosides.
包括MG-1655在内的大肠杆菌K12菌株，通过磷酸烯醇丙酮酸依赖性葡萄糖：磷酸转移酶系统（IICB(Glc)/IIA(Glc)）积累甲基-α-D-吡喃葡萄糖苷。高浓度的细胞内6-磷酸甲基-α-D-吡喃葡萄糖苷具有毒性，会阻止细胞生长。然而，用编码肺炎克雷伯菌aglB基因的质粒（pAP1）转化大肠杆菌MG-1655，使得转化体MG-1655（pAP1）在这种葡萄糖类似物上能良好生长。AglB是一种不同寻常的NAD⁺/Mn²⁺依赖性磷酸-α-葡萄糖苷酶，它通过催化体内6-磷酸甲基-α-D-吡喃葡萄糖苷水解生成6-磷酸葡萄糖和甲醇，从而促进MG-1655（pAP1）的生长。当用编码肺炎克雷伯菌基因aglB和aglA（一种α-葡萄糖苷特异性转运蛋白AglA（IICB(Agl)））的质粒pAP2转化时，菌株MG-1655（pAP2）能代谢多种其他α-连接的葡萄糖苷，包括麦芽糖醇、异麦芽糖以及蔗糖的以下五种异构体：海藻酮α(1→1)、松二糖α(1→3)、麦芽糖酮α(1→4)、异甜菊糖α(1→5)和帕拉金糖α(1→6)。值得注意的是，MG-1655（pAP2）不能代谢α(1→2)蔗糖。大肠杆菌K12菌株ZSC112L（ptsG::cat manXYZ nagE glk lac）既不能在葡萄糖上生长，也不能转运甲基-α-D-吡喃葡萄糖苷。然而，当用pTSGH11（编码ptsG）或pAP2转化时，该菌株分别提供了含有PtsG或AglA转运蛋白的膜。用纯化的通用磷酸转移酶组分对转运蛋白特异性膜进行体外互补实验表明，虽然PtsG和AglA都能识别葡萄糖和甲基-α-D-吡喃葡萄糖苷，但只有AglA能接受其他α-D-葡萄糖苷作为底物。互补实验还表明，IIA(Glc)是PtsG和AglA转运蛋白功能活性所必需的。我们得出结论，AglA、AglB和IIA(Glc)对于大肠杆菌K12在甲基-α-D-葡萄糖苷和相关α-D-吡喃葡萄糖苷上生长是必要且充分的。
